Ferulic Acid Esterase Activity Research Articles

Synergistic enzyme assisted release of ferulic acid from brewer´s spent grain

Brewer’s spent grain (BSG), the most abundant by-product of the beer industry, can be considered a sustainable source of ferulic acid (4-hydroxy-3-methoxycinnamic acid) (FA). FA is esterified to the arabinoxylan in the BSG. FA exhibits a variety of biological activities and is a target for valorization of BSG. We present a novel enzyme-assisted approach that utilizes the synergistic action of a specific bacterial endo-1,4-β-xylanase and a fungal ferulic acid esterase activity to selectively release high yields of FA from BSG. The study compares the efficacy of three different fungal feruloyl esterases (E.C. 3.1.1.73) of carbohydrate esterase family 1 (CE1), HiFae, AoFae1, and AnFae1, acting separately or in combination with two different endo-1,4-β-xylanases (EC 3.2.1.8), a bacterial GH30 enzyme from Dickeya chrysanthemi, and Shearzyme®, a commercial GH10 enzyme from Aspergillus aculeatus, respectively. On an equimolar dosage basis, the AnFae1-GH30 was the most efficient combination in releasing FA from BSG. This combination showed significant synergy in a dose-dependent manner, enabling up to 85% FA recovery from BSG within 30 min. of treatment. The results demonstrate a novel enzymatic approach for valorization of BSG as FA is considered a high-value natural compound for use in e.g. anti-aging skin care products.

Plant-Based Nutraceutical Formulation Modulates the Human Gut Microbiota and Ferulic Acid Esterase Activity During In Vitro Fermentation.

Oxidative stress is an imbalance between free reactive oxygen species and antioxidant defences leading to neurological and other chronic disorders. The interaction between food and gut microbiota and their metabolites significantly reduces oxidative stress and influences host physiology and metabolism. This process mainly involves enzymes that hydrolyse complex polysaccharides and produce metabolites. Ferulic acid esterases (FAE) one of the most important enzymes of the gut microbiome, release ferulic acid from feruloylated sugar ester conjugates, that occur naturally in grains, fruits, and vegetables. FA is crucial in combating oxidative stress resulted from free radical formation. This study investigated the effect of two plant-based nutraceutical formulations, cereal-millet-based (PC1) and fruit-vegetable-based (PC2), on gut microbiota and the production of FAE, short chain fatty acids (SCFA) and other small metabolites in in vitro fermentation using human faecal samples. After in vitro fermentation, both nutraceutical formulations increased the abundance of Bifidobacterium, Lactobacillus, Prevotella, Feacalibacteria, and Clostridium leptum. Furthermore, they induced the production of FAE, xylanase and pectinase enzymes, SCFA and other small metabolites, resulting in increased antioxidation activity of the fermentate. PC1 stimulated FAE and xylanase production more effectively. These results demonstrated a positive correlation between the feruloylated nutraceutical formulation and the production of FAE and other accessory enzymes, suggesting that PC1 and PC2 stimulate the proliferation of the FAE-producing microbial consortium of the gut microbiome and therefore, increase FA and SCFA concentration. From this study it is evident that FA-rich plant-based formulation can be used as a prophylactic nutraceutical supplement to alleviate oxidative stress by modulating the gut microbiota.

Degradation of ester linkages in rice straw components by Sphingobium species recovered from the sea bottom using a non-secretory tannase-family α/β hydrolase.

Microbial decomposition of allochthonous plant components imported into the aquatic environment is one of the vital steps of the carbon cycle on earth. To expand the knowledge of the biodegradation of complex plant materials in aquatic environments, we recovered a sunken wood from the bottom of Otsuchi Bay, situated in northeastern Japan in 2012. We isolated Sphingobium with high ferulic acid esterase activity. The strain, designated as OW59, grew on various aromatic compounds and sugars, occurring naturally in terrestrial plants. A genomic study of the strain suggested its role in degrading hemicelluloses. We identified a gene encoding a non-secretory tannase-family α/β hydrolase, which exhibited ferulic acid esterase activity. This enzyme shares the consensus catalytic triad (Ser-His-Asp) within the tannase family block X in the ESTHER database. The molecules, which had the same calculated elemental compositions, were produced consistently in both the enzymatic and microbial degradation of rice straw crude extracts. The non-secretory tannase-family α/β hydrolase activity may confer an important phenotypic feature on the strain to accelerate plant biomass degradation. Our study provides insights into the underlying biodegradation process of terrestrial plant polymers in aquatic environments.

Effects of Ferulic Acid Esterase-Producing Lactic Acid Bacteria and Storage Temperature on the Fermentation Quality, In Vitro Digestibility and Phenolic Acid Extraction Yields of Sorghum (Sorghum bicolor L.) Silage.

Two lactic acid bacteria (LAB) strains with different ferulic acid esterase (FAE) activities were isolated: Lactobacillus farciminis (LF18) and Lactobacillus plantarum (LP23). The effects of these strains on the fermentation quality, in vitro digestibility and phenolic acid extraction yields of sorghum (Sorghum bicolor L.) silage were studied at 20, 30 and 40 °C. Sorghum was ensiled with no additive (control), LF18 or LP23 for 45 days. At 40 °C, the lactic acid content decreased, whereas the ammonia nitrogen (NH3-N) content significantly increased (p < 0.05). At all three temperatures, the inoculants significantly improved the lactic acid contents and reduced the NH3-N contents (p < 0.05). Neither LP23 nor LF18 significantly improved the digestibility of sorghum silages (p > 0.05). The LP23 group exhibited higher phenolic acid extraction yields at 30 °C (p < 0.05), and the corresponding yields of the LF18 and control groups were improved at 40 °C (p < 0.05). FAE-producing LABs might partially ameliorate the negative effects of high temperature and improve the fermentation quality of sorghum silage. The screened FAE-producing LABs could be candidate strains for preserving sorghum silage at high temperature, and some further insights into the relationship between FAE-producing LABs and ensiling temperatures were obtained.

Application of Michaelis–Menten in the kinetics of oil palm frond enzymatic hydrolysis for ferulic acid production

Ferulic acid (FA) production has become a frequent focus on today’s research due to its antioxidant properties. However, there has been little to none studies reported on the usage of mixed culture as inoculum in FA production. This study aims to determine the kinetics FA production in a mixed culture fermentation by applying the Michaelis–Menten kinetic model. In this study, mixed culture fermentation process using oil palm frond bagasse (OPFB) as substrate was applied for FA production improvement. Optimum condition was applied to study ferulic acid esterase (FAE) mechanism for kinetic modelling purposes. The kinetic model used was based on the Michaelis–Menten kinetic model. Runge–Kutta Fourth Order method was used to solve the kinetic model. Maximum FAE activity was achieved at the 28 h of fermentation process at 3.7 × 10−3 mU mL−1. This result proved that enzymatic hydrolysis occurred during fermentation process. Kinetic study was conducted with Michaelis–Menten kinetic model used as a reference kinetic equation. Three kinetic constants, Vmax, Km and Ks were determined as 3.725 × 10−3 g L−1 h−1, 28.231 g L−1 and 1.33 × 10−2 h−1 respectively using Runge–Kutta Fourth Order approach. The outcome of this study confirms that the kinetics of the process fit well with the Michaelis–Menten model. This also suggests that the usage of mixed culture has the potential to induce enzymatic hydrolysis hence improving FA production from OPFB during fermentation process.

Simultaneous production of cellulase and ferulic acid esterase by Penicillium decumbens with rice straw as the sole carbon source

As well as cellulose and hemicelluloses, rice straw contains phenolic acids. The simultaneous production of monosaccharides and phenolic acids could improve the value of rice straw. In this study, it was confirmed that Penicillium decumbens produces more ferulic acid esterase (FAE) than other cellulase-producing fungi. Cellulose, destarched wheat bran (DSWB), and rice straw were used as carbon sources. Little phenolic acid was released by cellulose- and DSWB-based enzymes during the saccharification of rice straw, whereas rice straw was a favorable carbon source for the simultaneous production of cellulase and FAE. High-performance liquid chromatography showed that during enzyme production, phenolic acids were released from rice straw, and ball-milling affected this release of phenolic acids. Small amounts of phenolic acids induced FAE production. Although the enzymes produced with rice straw showed lower FAE activity than those produced with DSWB, phenolic acids were produced efficiently during the saccharification of rice straw in response to the synergistic effects of cellulase and FAE. Therefore, we suggest that the production of enzymes by P.decumbens on rice straw as the sole carbon source will allow the production of more valuable products from rice straw, making the utilization of rice straw more economic.

Characterization and identification of ferulic acid esterase‐producing Lactobacillus species isolated from Elymus nutans silage and their application in ensiled alfalfa

Ferulic acid esterase (FAE)-producing Lactobacillus species isolated from ensiled Elymus nutans growing on the Qinghai-Tibetan plateau were characterized, and effects of their application to the alfalfa ensiling process and the evidence to synergic effect between cellulase and FAE were investigated. The results of 16S rRNA gene sequence and species-specific polymerase chain reaction amplification showed that two screened strains with high FAE activity were Lactobacillus plantarum A1 (LP) and L. brevis A3 (LBr). The optimum temperature and pH for the LP and LBr was 37°C and 6·4 respectively. The FAE exhibited a good stability at temperatures between 25 and 50°C and at pH values of 5·0-7·0. The two strains and a commercial cellulase (CE) were applied as additives to alfalfa silage. After 60days of ensiling, the lactic acid in the control and CE groups were significantly lower than those of the other treatment groups. The neutral detergent fibre and acid detergent fibre contents in the LP group were significantly lower than those observed in the other groups. At the same time, the combination of CE and FAE-producing lactic acid bacteria synergistically improved the fermentation quality of the silage. The addition of the FAE-producing strain of L. plantarum A1 to alfalfa silage improved its fermentation quality, and reduced the fibre content of the silage. The screened homo-fermentative and FAE-producing strain of L. plantarum A1 could be a candidate strain in improving fermentation quality and fibre digestibility of ensiled forages.

Ferulic acid esterase-producing lactic acid bacteria and cellulase pretreatments of corn stalk silage at two different temperatures: Ensiling characteristics, carbohydrates composition and enzymatic saccharification

The effects of Acremonium cellulase and L. plantarum A1 with ferulic acid esterase activity on corn stalk silage fermentation characteristics, carbohydrate composition and enzymatic saccharification were studied at 25 and 40 °C, respectively. Corn stalk was ensiled without additive (C), Acremonium cellulase (AC), L. plantarum A1 (Lp) and AC + Lp for 60 days. Pretreatment with Lp or AC + Lp promoted the better silage fermentation and the degradation of lignocellulose as indicated by high lactic acid and low pH and lignocellulose content compared to control silages at 25 °C. AC + Lp performed better in reducing lignocellulose and DM loss. In addition, Lp alone enhanced enzymatic saccharification of corn stalk silage. However, the influence of L. plantarum A1 on corn stalk silage was not obvious at 40 °C. Corn stalk ensiled with combined additive is a suitable pretreatment method for subsequent biofuel production at 25 °C, but addition of Acremonium cellulase alone at 40 °C may be a promising method.

Production of Ferulic Acid Esterase from Aspergillus Oryzae (NCIM 1212)

The majority of valuable compounds used worldwide are produced by chemical synthesis at very low cost, though consumers prefer to natural compounds because of their health awareness in daily life. Ferulic acid is one of the most valuable compounds having widespread application in various fields such as in baking, cosmetic, beverage and pharma industries, also used as a raw material for the production of vanillin, the most flavouring compound. Ferulic acid esterase is the enzyme that hydrolyzes the ester bond between the ferulic acid and polysaccharides present in plant cell wall and release ferulic acid from agro residues. The production of ferulic acid esterase activity by strains was detected in agar plate assay. The assay involves ethyl ferulate as a sole carbon source in specific media. Of the examined strains Aspergillus oryzae (NCIM 1212) showed the highest level of ferulic acid esterase activity 281 U/mg at the optimum pH and temperature of 5.5 and 35°C. Supplementation of soyabean meal (0.75%) as nitrogen source and sucrose (0.1%) as additional carbon source favored enzyme production.

Crystal Structure and Substrate Specificity Modification of Acetyl Xylan Esterase from Aspergillus luchuensis.

Acetyl xylan esterase (AXE) catalyzes the hydrolysis of the acetyl bonds present in plant cell wall polysaccharides. Here, we determined the crystal structure of AXE from Aspergillus luchuensis (AlAXEA), providing the three-dimensional structure of an enzyme in the Esterase_phb family. AlAXEA shares its core α/β-hydrolase fold structure with esterases in other families, but it has an extended central β-sheet at both its ends and an extra loop. Structural comparison with a ferulic acid esterase (FAE) from Aspergillus niger indicated that AlAXEA has a conserved catalytic machinery: a catalytic triad (Ser119, His259, and Asp202) and an oxyanion hole (Cys40 and Ser120). Near the catalytic triad of AlAXEA, two aromatic residues (Tyr39 and Trp160) form small pockets at both sides. Homology models of fungal FAEs in the same Esterase_phb family have wide pockets at the corresponding sites because they have residues with smaller side chains (Pro, Ser, and Gly). Mutants with site-directed mutations at Tyr39 showed a substrate specificity similar to that of the wild-type enzyme, whereas those with mutations at Trp160 acquired an expanded substrate specificity. Interestingly, the Trp160 mutants acquired weak but significant type B-like FAE activity. Moreover, the engineered enzymes exhibited ferulic acid-releasing activity from wheat arabinoxylan.IMPORTANCE Hemicelluloses in the plant cell wall are often decorated by acetyl and ferulic acid groups. Therefore, complete and efficient degradation of plant polysaccharides requires the enzymes for cleaving the side chains of the polymer. Since the Esterase_phb family contains a wide array of fungal FAEs and AXEs from fungi and bacteria, our study will provide a structural basis for the molecular mechanism of these industrially relevant enzymes in biopolymer degradation. The structure of the Esterase_phb family also provides information for bacterial polyhydroxyalkanoate depolymerases that are involved in biodegradation of thermoplastic polymers.

Biochemical and Structural Analyses of Two Cryptic Esterases in Bacteroides intestinalis and their Synergistic Activities with Cognate Xylanases

Arabinoxylans are constituents of the human diet. Although not utilizable by the human host, they can be fermented by colonic bacteria. The arabinoxylan backbone is decorated with arabinose side chains that may be substituted with ferulic acid, thus limiting depolymerization to fermentable sugars. We investigated the polypeptides encoded by two genes upregulated during growth of the colonic bacterium Bacteroides intestinalis on wheat arabinoxylan. The recombinant proteins, designated BiFae1A and BiFae1B, were functionally assigned esterase activities. Both enzymes were active on acetylated substrates, although each showed a higher ferulic acid esterase activity on methyl-ferulate. BiFae1A showed a catalytic efficiency of 12mM s−1 on para-nitrophenyl-acetate, and on methyl-ferulate, the value was 27 times higher. BiFae1B showed low catalytic efficiencies for both substrates. Furthermore, the two enzymes released ferulic acid from various structural elements, and NMR spectroscopy indicated complete de-esterification of arabinoxylan oligosaccharides from wheat bran. BiFae1A is a tetramer based on the crystal structure, whereas BiFae1B is a dimer in solution based on size exclusion chromatography. The structure of BiFae1A was solved to 1.98Å resolution, and two tetramers were observed in the asymmetric unit. A flexible loop that may act as a hinge over the active site and likely coordinates critical interactions with the substrate was prominent in BiFae1A. Sequence alignments of the esterase domains in BiFae1B with the feruloyl esterase from Clostridium thermocellum suggest that both domains lack the flexible hinge in BiFae1A, an observation that may partly provide a molecular basis for the differences in activities in the two esterases.

Enrichment of maize and triticale bran with recombinant Aspergillus tubingensis ferulic acid esterase.

Ferulic acid is a natural antioxidant found in various plants and serves as a precursor for various fine chemicals, including the flavouring agent vanillin. However, expensive extraction methods have limited the commercial application of ferulic acid, in particular for the enrichment of food substrates. A recombinant Aspergillus tubingensis ferulic acid esterase Type A (FAEA) was expressed in Aspergillus niger D15#26 and purified with anion-exchange chromatography (3487U/mg, Km =0.43mM, Kcat=0.48/min on methyl ferulate). The 36-kDa AtFAEA protein showed maximum ferulic acid esterase activity at 50°C and pH 6, suggesting potential application in industrial processes. A crude AtFAEA preparation extracted 26.56 and 8.86mg/g ferulic acid from maize bran and triticale bran, respectively, and also significantly increased the levels of p-coumaric and caffeic acid from triticale bran. The cost-effective production of AtFAEA could therefore allow for the enrichment of brans generally used as food and fodder, or for the production of fine chemicals (such as ferulic and p-coumaric acid) from plant substrates. The potential for larger-scale production of AtFAEA was demonstrated with the A. niger D15[AtfaeA] strain yielding a higher enzyme activity (185.14 vs. 83.48U/ml) and volumetric productivity (3.86 vs. 1.74U/ml/h) in fed-batch than batch fermentation.

Effects of Two-stage Controlled pH and Temperature vs. One-step Process for Hemicellulase Biosynthesis and Feruloyl Oligosaccharide Fermentation using Aureobasidium pullulans

A two-stage, pH- and temperature-controlled wheat bran fermentation method using Aureobasidium pullulans was investigated for feruloyl oligosaccharides (FOs) production and the activities of xylanase, xylosidase, and ferulic acid esterase (FAE). A. pullulans secreted xylanase, xylosidase, and FAE at high levels in the initial pH of 4.0 to 5.0 and a fermentation liquid temperature of 31 °C to 33 °C. FOs production via two-stage fermentation (FOs 2) reached 1123 nmol/L after fermentation for 96 h, by controlling the initial pH at 4.0 and the initial temperature at 33 °C, and then changing the pH to 6.0 and the temperature to 29 °C at the same time at 36 h. This process was 12 h shorter and 219 nmol/L higher than a one-stage fermentation for producing FOs 1. Xylanase, xylosidase, and FAE activities were highly correlated with controlled pH and temperature and FOs biosynthesis rate. Thus, the combination of two-stage controlled pH and temperature could support mass production of FOs.

Contributions of a unique β-clamp to substrate recognition illuminates the molecular basis of exolysis in ferulic acid esterases.

Lignocellulosic biomass is a promising renewable resource; however, deconstruction of this material is still the rate-limiting step. Major obstacles in the biocatalytic turnover of lignocellulose are ester-linked decorations that prevent access to primary structural polysaccharides. Enzymes targeting these esters represent promising biotools for increasing bioconversion efficiency. Ruminant livestock are unique in their ability to degrade lignocellulose through the action of their gut microbiome. The anaerobic fungi (phylum Neocallimastigomycota) are key members of this ecosystem that express a large repertoire of carbohydrate-active enzymes (CAZymes) with little sequence identity with characterized CAZymes [Lombard, Golaconda, Drula, Coutinho and Henrissat (2014) Nucleic Acids Res. 42: , D490-D495]. We have identified a carbohydrate esterase family 1 (CE1) ferulic acid esterase (FAE) belonging to Anaeromyces mucronatus(AmCE1/Fae1a), and determined its X-ray structure in both the presence [1.55 Å (1 Å=0.1 nm)] and absence (1.60 Å) of ferulic acid. AmCE1 adopts an α/β-hydrolase fold that is structurally conserved with bacterial FAEs, and possesses a unique loop, termed the β-clamp, that encloses the ligand. Isothermal titration calorimetry reveals that substrate binding is driven by enthalpic contributions, which overcomes a large entropic penalty. A comparative analysis of AmCE1 with related enzymes has uncovered the apparent structural basis for differential FAE activities targeting cross-linking ferulic acid conjugates compared with terminal decorations. Based on comparisons to structurally characterized FAEs, we propose that the β-clamp may define the structural basis of exolytic activities in FAEs. This provides a structure-based tool for predicting exolysis and endolysis in CE1. These insights hold promise for rationally identifying enzymes tailored for bioconversion of biomass with variations in cell wall composition.

Ferulic Acid Produced by Lactobacillus fermentum NCIMB 5221 Reduces Symptoms of Metabolic Syndrome in Drosophila melanogaster

Abstract The gut microbiota is an intricate community of thousands of bacterial species living in the gastrointestinal tract critical for vitamin synthesis, mineral absorption, digestion of otherwise indigestible fibers and the extraction of energy from food. Recently, the health of the gut microbiota’s community architecture has become linked to energy-regulatory diseases including metabolic syndrome: a collection of symptoms including hyperglycemia, hypercholesterolemia, high blood pressure, increased abdominal fat and triglyceride levels. However, the mechanism of communication between the gut microbiota and the host energy metabolism remains elusive. The current study shows that ferulic acid (FA) produced by the intrinsic ferulic acid esterase activity of the probiotic bacteria Lactobacillus fermentum NCIMB 5221 (Lf5221) can dose-dependently rescue the phenotypic markers of diet-induced diabetes and obesity in Drosophila melanogaster. In Drosophila exposed to either a high-sugar or high-fat diet, living Lf5221 at 2.5 or 7.5 × 109 CFU/ml media effectively rescued whole-body weight, glucose, trehalose and triglyceride levels. All of the aforementioned effects were lost in heat-inactivated bacteria indicating that a metabolic product is responsible. Likewise, FA at 0.5 mM in the metabolically challenged Drosophila models reared similar effects on the physiological markers while also reducing hyperglycemia in the circulating hemolymph. On the signaling level, the high-sugar diet predictably had an elevated expression of the Drosophila insulin-like peptides 2, 3 and 5 and in the high-fat diet, an increase in fatty acid synthase, acetyl-CoA carboxylase and phosphoenolpyruvate carboxykinase expression. On both diets, Lf5221 and FA rescued gene expression, at different concentrations, to the level of controls. Examining the mechanistic gene expression, both Lf5221 and FA rescued expression of dFOXO and dTOR, but not dAkt indicating that FA produced by Lf5221 is acting on one of the downstream-signaling molecules from the insulin receptor, possibly dTOR: an overall energy regulator in flies and humans alike. The present study for the first time outlines a streamlined mechanism for how the gut microbiota communicates with the host’s energy-regulating metabolism. Proper supplementation with ferulic acid esterase active probiotics such as Lf5221 could potentially prevent or alleviate symptoms of metabolic syndrome and other energy-regulating diseases including diabetes, obesity and neurodegeneration.

An unusual feruloyl esterase belonging to family VIII esterases and displaying a broad substrate range

A thermophilic compost metagenomic library constructed in Escherichia coli was functionally screened for novel esterases. Of the 110,592 fosmid clones screened, 25 clones demonstrated degradative activity on glyceryl tributyrate (a hit rate of 1:4,423). Four clones displayed ferulic acid esterase activity and were sequenced using 454 Titanium sequencing technology. EstG34, a 410 amino acid protein, was identified as having high sequence identity with a number of bacterial β-lactamases. EstG34 has the S-X-X-K motif which is conserved in class C β-lactamases and family VIII carboxylesterases. Purified recombinant EstG34 had a molecular mass of 42kDa and displayed hydrolytic activity towards a variety of p-nitrophenyl esters, hydroxycinnamic acid esters and α-naphthol acetate. EstG34 represents the first family VIII carboxylesterase and β-lactamase fold enzyme, able to hydrolyse ferulate and a number of other hydroxycinnamic acid esters. In addition, EstG34 is the first reported FAE to not adopt the α/β hydrolase conformation. The sequence similarity and wide substrate utilization capability of this esterase complicates its placement within current classification systems, but also draws attention to the enzyme's potential versatility.

Functional co-expression of a fungal ferulic acid esterase and a β-1,4 endoxylanase in Festuca arundinacea (tall fescue) modifies post-harvest cell wall deconstruction.

Improved post-harvest cell wall deconstruction of tall fescue leaves has been demonstrated by in-planta co-expression of a constitutively expressed ferulic acid esterase together with a senescence-induced β-1,4 endoxylanase. Tall fescue plants (Festuca arundinacea) constitutively expressing vacuole- or apoplast-targeted ferulic acid esterase from Aspergillus niger were retransformed with a senescence-induced and apoplast-targeted β-1,4 endo-xylanase from Trichoderma reesei. Enzyme activities in co-expressing plants stabilized after repeated vegetative propagation, with xylanase activity in senescent leaves increasing and ferulic acid esterase activity decreasing after tillering. Plants co-expressing both enzymes in the apoplast, with the lowest levels of ferulate monomers and dimers and the lowest levels of cell wall arabinoxylans, released ten times more cell wall hydroxycinnamic acids and five times more arabinoxylan from the cell wall on autodigestion compared to expression of ferulic acid esterase or xylanase alone. These plants also showed a 31 % increase in cellulase-mediated release of reducing sugars, a 5 % point increase in in vitro dry matter digestibility and a 23 % increase in acetyl bromide-soluble lignin. However, plant growth was adversely affected by expressing FAE in the apoplast, giving plants with narrower shorted leaves, and a 71 % decrease in biomass.

Effect of orally administered L. fermentum NCIMB 5221 on markers of metabolic syndrome: an in vivo analysis using ZDF rats

Metabolic syndrome, encompassing type 2 diabetes mellitus and cardiovascular disease, is a growing health concern of industrialized countries. Ferulic acid (FA) is a phenolic acid found in foods normally consumed by humans that has demonstrated antioxidant activity, cholesterol-lowering capabilities, and anti-tumorigenic properties. Select probiotic bacteria, including Lactobacillus fermentum NCIMB 5221, produce FA due to intrinsic ferulic acid esterase activity. The aim of the present research was to investigate a FA-producing probiotic, L. fermentum NCIMB 5221, as a biotherapeutic for metabolic syndrome. The probiotic formulation was administered daily for 8 weeks to Zucker diabetic fatty (ZDF) rats, a model of hyperlipidemia and hyperglycemia. Results show that the probiotic formulation reduced fasting insulin levels and insulin resistance, significantly reduced serum triglycerides (p = 0.016), lowered serum low-density lipoprotein cholesterol levels (p = 0.008), and significantly reduced the atherogenic (p = 0.016) and atherosclerosis (p = 0.012) index as compared to the control animals. In addition, the probiotic formulation significantly increased high-density lipoprotein cholesterol levels (p = 0.041) as compared to the control animals. This research indicates that administration of the FA-producing L. fermentum NCIMB 5221 has the potential to reduce insulin resistance, hyperinsulinemia, hypercholesterolemia, and other markers involved in the pathogenesis of metabolic syndrome. Further studies are required to investigate the human clinical potential of the probiotic formulation in affecting the markers and pathogenesis of metabolic syndrome.

Biotransformation of Rice Bran to Ferulic Acid by Pediococcal Isolates

Ferulic acid (FA) is widely used in foods, in beverages, and in various pharmaceutical industries as a precursor of vanillin. FA biotransformation can occur during the growth of lactic acid bacteria (LAB), and its conversion to other phenolic derivatives is observed by many scientists, where ferulic acid esterase (FAE) and ferulic acid decarboxylase (FDC) play significant roles. The present study aimed at screening a panel of LAB for their ability to release FA from rice bran, an agro waste material. FAE and FDC activities were analyzed for the preliminary screening of various dairy isolates. Two Pediococcus acidilactici isolates were selected for studying further the hydrolysis of FA from rice bran and its bioconversion into phenolic derivatives like 4-ethylphenol, vanillin, vanillic acid, and vanillyl alcohol. P. acidilactici M16, a probiotic isolate, has great potential for the production of FA from rice bran and could be exploited as starter culture in the food industry for the production of biovanillin.

Enzymatic characteristics of crude feruloyl and acetyl esterases of rumen fungus Neocallimastix sp. YAK11 isolated from yak (Bos grunniens)

Rumen fungus Neocallimastix sp. YAK11 was isolated from yak (Bos grunniens), and three consecutive 10-day pure cultures were anaerobically performed at 39 °C in 20-ml Hungate's tubes to explore ferulic acid esterase (FAE) and acetyl esterase (AE) activity profiles of the fungus grown on whole hay fraction of Chinese wildrye grass (Leymus chinensis) (WHOcw , n = 4) and its neutral detergent fibre fraction (NDFcw , n = 4), respectively. An aliquot of 0.7-ml culture was sampled daily using a sterile syringe, and 0.7-ml fresh medium was immediately added to the tubes to compensate for the withdrawn samples. Peak esterase activity occurred for FAE on day 5 (p < 0.001) and for AE on day 6 (p < 0.001). The mean activities of FAE and AE in WHOcw were 2.07 and 1.29 times of those in NDFcw (p < 0.001). Both FAE and AE activities were positively correlated with xylanase (r > 0.65, p < 0.001) and carboxymethyl cellulase (r > 0.57, p < 0.001) activities. Total volatile fatty acid concentration was positively correlated with enzyme activities of AE (r > 0.87, p < 0.001), FAE (r > 0.82, p < 0.001) and xylanase (r > 0.56, p < 0.001). Crude enzyme solution was harvested for the fungus grown on WHOcw , and the pH optimum of FAE activity was 8.0 while the optimum for AE was 9.0. Both FAE and AE had a broad pH stability range. The optimal temperatures for FAE and AE activity were 40 and 50 °C. The Michaelis constant (Km ) and maximum velocity (Vmax ) for FAE against methyl ferulate at pH 6.0 and 39 °C were 0.078 mm and 2.93 mU, respectively. The Km and Vmax for AE against p-nitrophenyl acetate at pH 7.0 and 39 °C were 2.73 mm and 666.67 mU, respectively. Both FAE and AE may have prospective advantages for the enzymatic degradation of roughages in ruminant animals.